Site-directed mutagenesis of bifunctional riboflavin kinase/FMN adenylyltransferase via CRISPR/Cas9 to enhance riboflavin production

Vitamin B2 is an essential water-soluble vitamin. For most prokaryotes, a bifunctional enzyme called FAD synthase catalyzes the successive conversion of riboflavin to FMN and FAD. In this study, the plasmid pNEW-AZ containing six key genes for the riboflavin synthesis was transformed into strain R2 with the deleted FMN riboswitch, yielding strain R5. The R5 strain could produce 540.23 ± 5.40 mg/L riboflavin, which was 10.61 % higher than the R4 strain containing plasmids pET-AE and pAC-Z harboring six key genes. To further enhance the production of riboflavin, homology matching and molecular docking were performed to identify key amino acid residues of FAD synthase. Nine point mutation sites were identified. By comparing riboflavin kinase activity, mutations of T203D and N210D, which respectively decreased by 29.90 % and 89.32 % compared to wild-type FAD synthase, were selected for CRISPR/Cas9 gene editing of the genome, generating engineered strains R203 and R210. pNEW-AZ was transformed into R203, generating R6. R6 produced 657.38 ± 47.48 mg/L riboflavin, a 21.69 % increase compared to R5. This study contributes to the high production of riboflavin in recombinant E. coli BL21.


Introduction
Vitamin B 2 , also known as riboflavin (RF), is an essential watersoluble vitamin, and it was first isolated from milk in the late 1870s [1].Currently, RF is widely used in the pharmaceutical, feed, cosmetic, and food industries [2].The derivates of RF, flavin mononucleotide (FMN) [3] and flavin adenine dinucleotide (FAD) [4], are important cofactors for oxidoreductases and dehydrogenases in almost all organisms [5], and are involved in oxidative metabolism, energy metabolism, vitamin metabolism, and other processes [6,7].RF deficiency can interfere with the maintenance of reduced glutathione, cause cellular stress, generate nonfunctional proteins, and subsequently cause many diseases [1,8,9].
In recent decades, some genetically modified strains, including Bacillus subtilis, Corynebacterium ammoniagenes, Candida spp.and Ashbya gossypii, were constructed for the RF biosynthesis through different strategies [2,[10][11][12][13][14][15].Although wild-type Escherichia coli cannot accumulate RF in common conditions, it is still a promising RF-producing bacterium due to its rapid growth, clear genetic background, ease of metabolic modification, high expression of recombinant proteins and low yield of acetic acid [16].In addition, compared to other E. coli strains, E. coli BL21 can accumulate RF under natural conditions because it has a His115Leu mutation in FAD synthase (FADS, encoded by ribF), causing a decrease in RibF enzyme activity and an increase in the expression of RF synthesis genes.
RF is first catalyzed to FMN, and then to FAD in E. coli BL21 by a bifunctional enzyme FADS (Fig. 1).FADS folds into two nearly independent modules, the C-terminus with riboflavin kinase (RFK) activity and the N-terminus with FMN aminotransferase (FMNAT) activity [9,17].To increase RF production, it is a key point to reduce its conversion to FMN and FAD.Since ribF is an essential gene for the growth of E. coli [18][19][20], its knockout is lethal to the strain.Therefore, we can only reduce the enzymatic activity of FADS by some means to enhance the production of RF.A variety of metabolic engineering approaches have been applied to down-regulate FADS activity, and these operations have improved the production of RF in different microorganisms [13,21,22].However, no information can be obtained on enhancing RF production by down-regulating FADS activity via site-directed mutagenesis in E. coli BL21.
In this study, a 4-isopropylbenzoicacid (cumate)-inducible plasmid pNEW-AZ containing six key genes in RF synthesis of E. coli, ribA, ribB, ribC, ribD, ribE and zwf, was constructed for facilitating the production of RF.Homology matching and molecular modeling were used to determine the potential mutated sites of FADS, and pETDuet-1 was employed to express mutated FADS.CRISPR/Cas9 was performed to complete the site-directed mutation of ribF in the E. coli BL21(DE3) genome to generate strain R203.The pNEW-AZ plasmid was transformed into strain R203 to obtain strain R6.Through these efforts, the titer of RF was enhanced to 657.38 ± 47.48 mg/L with 10 g/L glucose.

Plasmids, primers, strains, media, and growth conditions
All plasmids and strains used in this study are listed in Table 1.All primers used in this study are listed in Table 2. E. coli DH5α was used as the host to propagate plasmid DNA, and E. coli BL21(DE3) was used as an expression host.The expression plasmid pETDuet-1 purchased from Novagen Co. Ltd, USA, was employed to construct mutated ribF and express the mutant proteins.The expression plasmid pNEW, which was purchased from FORHIGH BIOTECH, was used to express key genes of RF synthesis.
Luria-Bertani (LB) medium (tryptone 10 g/L, yeast extract 5 g/L, and NaCl 10 g/L) was used for strain propagation and screening of RFproducing strains.Appropriate antibiotics were added to LB when necessary.Unless specifically mentioned, all the strains were incubated in LB at 37 • C under good aeration.
The FastPure plasmid mini kit, FastPure gel DNA extraction mini kit, FastPure® bacteria DNA isolation mini kit, and ClonExpress II one-step cloning kit were purchased from Vazyme Biotech (Nanjing, China).PrimeSTAR® HS (Premix), the competent cell preparation kit and restriction endonucleases (BamH I, Not I, Kpn I, Nhe I and Dpn I) were purchased from Takara Bio (Dalian, China).Ni-NTA His Bind Resin was purchased from Sangon Biotech (Shanghai, China).The BCA protein assay kit, SDS-PAGE preparation kit, and standard samples (RF & FMN & FAD) were purchased from Shanghai Biotech (Shanghai, China).Primer synthesis and sequencing were performed to Shanghai Biotech, China.

Construction of expression plasmids
The plasmids pET-AE and pAC-Z harboring the nucleotide fragments ribC-ribE-ribB-ribD-ribA and zwf for the synthesis of RF were constructed before in our laboratory [23].To construct an expression plasmid with cumate as an inducer, primers AE-F/R were used to amplify pET-AE to obtain the gene fragment ribC-ribE-ribB-ribD-ribA.Meanwhile, Z-F/R was used to amplify the plasmid pAC-Z to obtain the nucleotide fragment zwf.These two fragments were ligated by overlap PCR with the primers AE-F/Z-R, and the products were extracted and homologously recombined with a linear fragment of pNEW that had been previously double digested by Kpn I and Nhe I (Supplementary Fig. S1 a).
To obtain the plasmid to express RibF, the BL21 (DE3) genome DNA was extracted using the FastPure® bacteria DNA isolation mini kit and used as a PCR template and amplified with ribF-F/R as the primers.The PCR products were purified by a FastPure Gel DNA extraction mini kit, and homologous recombination was performed to insert the ribF gene fragment into pETDuet-1 (digested by BamH I & Not I) by a ClonExpress II one-step cloning kit, named pET-ribF (Supplementary Fig. S1 b).Reserve PCR was performed to obtain mutated ribF with pET-ribF as a template, and the PCR products were transformed into E. coli DH5α competent cells after digestion by Dpn I. Positive colonies were screened out by sequencing and stocked in glycerol tubes at − 80 • C. The constructed plasmids were respectively transformed into E. coli cells by heat shock except for CRISPR gene editing.

Preparation of cytosolic fractions and SDS-PAGE analysis of the target proteins
The expression plasmids were transformed into E. coli BL21(DE3) competent cells to express wild-type or mutated RibF.Recombinant E. coli BL21 (DE3) was incubated in 50 mL LB medium containing 100 μg/mL ampicillin at 37 • C. When the OD 600 value reached 0.6-0.8,isopropyl β-D-thiogalactopyranoside (IPTG) with a final concentration of 100 mM was added into LB medium to induce the expression of target proteins at 28 • C overnight.The cells were collected by centrifugation at 13,400×g for 10 min, and after removing the supernatant, the obtained cells were resuspended in 1 × PBS buffer (pH 7.4).Proteins were released by ultrasonic treatment (SCIENTZ-II D, China): work 2 s, pause 5 s, 150 W, 10 min.The resulting crushing solution was centrifuged (13,400×g, 10 min, 4 • C), and the pellet was further resuspended in 1 × PBS buffer.
To extract the crude proteins from mutated strains, single colonies of mutated strains were incubated with 5 mL LB medium overnight at 220 rpm and 37 • C. The cultures were added to 100 mL LB medium.When the OD 600 value reached ~1.0, the cells were collected by  centrifugation.The crushing procedure was described above.The supernatants were stored in a − 80 • C refrigerator for the subsequent analysis.
SDS-PAGE (12 % separating gel and 5 % stacking gel) was performed to identify the expression of target proteins.Coomassie brilliant blue G-250 was used to visualize the resolved proteins.

Purification of target proteins
For purification of the target proteins, Ni 2+ -NTA column chromatography was performed.The supernatants prepared from each cell culture were applied to a Ni 2+ -Sepharose column equilibrated with binding/washing buffer (20 mM Tris-HCl, 500 mM NaCl, 1 mM PMSF, and 30 mM imidazole, pH 7.4) at a flow rate of 1 mL/min.Subsequently, the column was washed with the same buffer to remove other proteins, and the histidine-tagged proteins were eluted with elution buffer (20 mM Tris-HCl, 500 mM NaCl, and 300 mM imidazole, pH 7.4).
The concentrations of the purified proteins were determined using the BCA kit, following the manufacturer's procedures.

Detection of quaternary organizations in vitro
There is no evidence as to whether FADS from E. coli (EcFADS) also forms a dimer-trimer conformation [17].To determine the potential assembly of EcFADS in solution, a protein crosslinking reaction was performed.Samples containing 30 ng of EcFADS (42 μM, 20 μL) in 20 mM PIPES, pH 7.0, were incubated for 30 min at 25 • C in both the absence and presence of 3.5 μM bis(sulfosuccinimidyl)suberate (BS 3 ) crosslinker (Aladdin, China).The crosslinking reaction was stopped by the addition of 0.5 M Tris/HCl, pH 8.0, until a final concentration of 50 mM [24].Reaction products were then resolved by SDS-PAGE (15 % separating gel and 5 % stacking gel).

Sequence alignment and docking simulations
ClustalW was used to align four FADS amino acid sequences derived from C. ammoniagenes, Mycobacterium tuberculosis, E. coli BL21(DE3), and B. subtilis.The results of the comparison were downloaded, and then make a graph using ESPript 3.0.For docking simulations, the structure model of EcFADS was built via swiss-model [https://swissmodel.expasy.org/] by using the D298E mutant of FAD synthetase from C. ammoniagenes (SMTL ID: 5fo0.1 [https://swissmodel.expasy.org/templates/5fo0]) as a template.The Autodock Vina software was used to simulate the binding of the EcFADS model to the substrate RF.An AMBER force-field based algorithm was used to accurately forecast the free energy of binding.The ligands final conformations were chosen based on the estimated binding energies.The following docking box settings were configured when using the local docking mode: The coordinates of the center were as follows: center x = − 3.785, center_y = − 32.715, and center_z = − 25.368.The dimensions of the object are: size_x = 62, size_y = 60, and size_z = 56, with grid spacing of 0.375 Å, and forecasts of 20 poses.The Ligplot software was used to view the docking results.The active central sites and conserved amino acid sites were identified based on the results of molecular docking and homology matching.

Measurement of the activity of RFK
As RF is successively converted to FMN and FAD via ribF-encoded RFK and FMNAT, reducing RFK activity decreases the consumption of RF.Thus, RFK activity was measured to compare the effect of different mutants.
Before detecting the activity of RFK, the concentration of purified proteins was measured by the BCA kit and using bovine serum albumin as a standard.RFK activity was measured in a final volume of 1 mL of potassium phosphate buffer (pH 7.5, containing 50 μM RF, 3 mM ATP, 15 mM MgCl 2 , and 10 mM Na 2 SO 3 ).The mixture was preincubated at 37 • C for 5 min, and 20-60 nM enzymes (higher concentrations used for variants with very low activities) [4] were added into the mixture to initiate the reaction.The mixture was then incubated at 37 • C for 10 min and then heated at 100 • C for 5 min to stop the reaction [4,25].A total of 63 μg crude enzymes were added to the mixture as described above when measuring the RFK activity of the mutant strains, and the reaction time was prolonged to 30 min.Centrifugation was performed to remove the proteins, and the FMN concentration in the supernatant was analyzed by HPLC.
To further determine the steady-state rates of WT EcFADS and mutants, the RFK activity was measured at increasing concentrations of RF (1-50 μM) and a saturated concentration of ATP, and at increasing concentrations of ATP (0.01-0.8 mM) and a saturated concentration of RF [26].The data obtained were fitted to the Michaelis-Menten equation to obtain Michaelis-Menten constant (Km) and catalytic rate (kcat) [26].

CRISPR/Cas9 genomic editing
CRISPR/Cas9 was carried out to achieve the replacement of native ribF with mutated ribF in the E. coli BL21(DE3) genome.RibF is an essential gene for E. coli [20] and avoids cleavage of the repair template by pCas due to the small difference between the inserted gene and the native gene.Two-step genomic editing was performed.First, the ribF gene on the E. coli BL21(DE3) genome was replaced by a fragment

Table 2
Primers used in this study.consisting of ribC from B. subtilis (encoding a flavonase/FAD-synthase) and a 63 bp sequence immediately (GTAGTAAAGGCGCTTCAATCAT-GAACATA ACTCAATT TGTAGGGTCATAGTAATCCAGCAACT) followed by ribC termination codon TAA.The fragment was synthesized by Sangon Biotech and named ribC opt .This fragment was inserted into pET-3a (+), generating pET-3a(+)-ribC opt .Second, ribC opt was replaced by the appropriate mutated ribF to generate engineered strains.The procedure of CRISPR/Cas9 was described in detail [27].sgRNA-ribF was obtained by reverse PCR with sgRNA-ribF-F/R as primers and pTargetF as the template.PCR products were treated with Dpn I and then transformed into E. coli DH5α competent cells.After heat shock and incubation, the culture was spread on LB plates with 50 μg/L spectinomycin for incubation at 30 pCas was transformed into E. coli BL21(DE3)-ΔsroG (R2) [23] to generate R2-pCas.Electroporation-competent R2-pCas cells were produced as described previously [23].Then, 400 ng sgRNA-ribF and 2.0 μg donor DNA were added to 100 μL electroporation-competent cells, and after 30 min of incubation on ice, electroporation was performed.After 1.5 h incubation with 900 μL LB medium without antibiotic at 30 • C and 140 rpm, the culture was spread on LB plates with 50 μg/L spectinomycin and 50 μg/L kanamycin and incubated overnight at 30 • C. Positive clones were screened by colony PCR and further verified by sequencing.
After curing the sgRNA-ribF with 0.5 mM IPTG, the positive colony containing pCas was subjected to the next operation.sgRNA-ribC opt which targets the 63 bp sequence was obtained by reverse PCR, with pTargetF as the template and sg-ribC opt -F/R as primers.The primers Up (F)-F/R and Down(F)-F/R were used to amplify E. coli BL21 (DE3) genomic DNA, and ~500 bp upstream and downstream homologation arms were obtained, named Up(F) and Down(F).The intermediate nucleic acid fragment was obtained by amplifying a suitable mutant ribF-containing plasmid with primers Mid(F)-F/R.The three fragments were amplified with primers Up(F)-F/Down(F)-R to obtain donor DNA.The subsequent electroporation process was carried out as described previously.Subsequently, sgRNA-ribC opt and pCas were sequentially cured by IPTG induction and 42 • C incubation, respectively.

Transformation of the expression plasmids
Single colonies of E. coli BL21(DE3) gene-edited bacteria were picked and incubated in antibiotic-free LB medium to an OD 600 value between 0.35 and 0.6.Competent cells of E. coli BL21(DE3) gene editing bacteria were prepared on ice using the competent cell preparation kit (Takara).The expression plasmids were transformed into competent cells by heat shock, and a single colony was used for RF production.

Statistical analysis
All experiments were performed in triplicate, and the results are presented as the means ± SDs.Statistical analysis was performed by SPSS 17.0 software.Data were graphed using Origin 8.5 software.A plasmid construction schematic map was drawn with ChemDraw Professional 2017 software.

Optimization of the concentration of the inducer cumate
It is generally accepted that the pET series vectors can only be expressed in E. coli BL21(DE3) hosts containing T7 RNA polymerase.However, in this study, when the expression plasmid pET-AE was transformed into E. coli DH5α, a certain amount of RF was detected in the fermentation broth of recombinant strain even in the absence of the inducer IPTG (~20 mg/L RF), indicating that the specificity of the T7 promoter is not ideal.Furthermore, the leaky expression of the pET series vectors severely compromised the expression of target genes [30], as the presence of flavin produced severe oxidative stress [31].For these reasons, we chose to replace the pET series vector with the pNEW vector, which has the same expression intensity as the pET vector series, is more tightly regulated and has a cheaper induction agent (cumate) [32].
After successful construction of pNEW-AZ, the plasmid was transformed into R2 to generate strain R5.To investigate the effect of RF production at different concentrations of cumate, the engineered strain R5 was incubated in 5 mL LB medium (with 50 mg/L kanamycin).After overnight incubation, 2 % (v/v) culture was added to 50 mL LB medium in a 250 mL shake flask.When the OD 600 value reached ~0.6-0.8, 1, 5, 10, 25, 50, 100, 200, and 500 μM cumate was added to 50 mL LB medium to induce the expression of target genes.
As shown in Fig. 2, after 72 h of incubation, the highest RF titer was 540.23 ± 5.40 mg/L with an induction concentration of 100 μM (with a yield of 59.42 ± 0.59 mg RF/g glucose), which was 10.61 % higher than that of strain R4 (produced 488.40 ± 11.18 mg/L RF in LB medium) [23].The increase in RF production may not only be due to reduced leakage expression, but also to a reduction in the number of plasmids and thus a lower metabolic burden.When cumate concentrations were between 1 and 10 μM, RF titers showed a linear increase with little difference in OD 600 values.When cumate concentrations were between 10 and 100 μM, the RF titers increased further with increasing inducer concentrations.The high concentrations of inducer (≥500 μM) were not effective in raising the RF titer.Because cumate is an acidic compound, too high concentration will lower the pH of the medium, which in turn will affect the growth of the strain.

Protein cross-linking experiments and determination of RibF mutation sites
FADS amino acid sequences derived from C. ammoniagenes, M. tuberculosis, E. coli BL21(DE3), and B. subtilis were aligned, and a conserved sequence GFPTAN (205-210, E. coli numbering) was observed (Fig. 3a), in which PTAN (207-210) is proposed to stabilize the metal ion and the phosphate groups of the ATP: Mg 2+ substrate during RFK catalysis [4].FADS from C. ammoniagenes (CaFADS) mainly stabilizes in hexameric assemblies in solution in a dimer-of-trimers conformation [9].In this study, EcFADS was found to be presented in solution as a monomer (trace amounts of multimeric forms may be present) by protein cross-linking experiments in vitro (Fig. 4).Although there are differences in the way these two FADS are assembled in solution, the system still recommends CaFADS as a template for molecular docking, and the binding energy of EcFADS co-docked to the substrates ATP and RF was − 9.825 kcal/mol.A total of fourteen amino acids were identified in the active center bound to ATP, of which four amino acids (D198, T247, E256, and H258) formed hydrogen bonding interactions and ten amino acids (P207, V223, V248, A249, G250, E282, F285, L288, L291, and I295) formed hydrophobic interactions (Fig. 3b and c).The molecular docking results of substrate RF showed that the following eight amino acids were present around the active site, in which four amino acids form hydrophobic interactions (T203, H258, L260, and R303), and the other four form hydrogen bonding interactions (I204, L259, D261, and E299) (Fig. 3b and c).Due to time constraints, this study tried to reduce RFK enzyme activity by mutating key amino acids that interact with the substrate RF.Ultimately, the mutant sites T203A, T203D, I204D, N210D, H258A, H258D, L259D, L260A, D261A, E299 N, R303A, and R303D were determined.

Expression and purification of the RibF variants and detection of RFK activity
The above strains were used to induce the expression of the target proteins by IPTG, and the supernatants after cell crushing were analyzed by SDS-PAGE.According to SDS-PAGE analysis (Supplementary Fig. S2), target proteins with a molecular weight of 35 kDa were obtained in all strains, which was consistent with the expected value, indicating that proteins were correctly expressed in all thirteen strains.Target proteins were successfully purified from all strains by Ni 2+ -NTA columns, except for strain B259 (Supplementary Fig. S3).The mutation at L259D may have caused a dramatic change in spatial structure, allowing 6 × His to be encapsulated.Therefore, eleven different purified mutant RibF proteins were used for comparison of RFK activity.
Detection of RFK activity was carried out with a fluorescence detector.The RFK activity of different mutated proteins is shown in Table 3.The single amino acid mutations that caused complete loss of enzyme activity were I204D, H258A/D, and R303D.Compared to WT RibF, the RFK enzyme activities of the three RibF mutated proteins, Fig. 2. production and OD 600 of the R5 strain at different cumate concentrations.The hydrogen bonds between the amino acid and the substrate are represented with dotted lines (green).The hydrophobic bonds are represented with an eyelash shape.c.A three-dimensional diagram of the molecular docking results of substrates interaction with the RFK module of WT EcFADS.RF and ATP ligands are shown as sticks and colored with carbons in green and yellow, respectively.The Mg 2+ cation is shown as a yellow sphere.The FMNAT module is blue and the RFK module is pink.T203D, N210D and R303A, were decreased by 29.90 %, 89.32 % and 15.92 %, respectively.The other three mutated proteins (L260A, D261A and E299 N) showed negligible changes in the RFK enzyme activity, with decreases by 2.14 %, 1.94 %, and 1.55 %, respectively.Surprisingly, the RFK enzyme activity of the T203A mutant protein was increased by 103.11 %.
As ribF is an essential gene, the mutation site with the greatest reduction in RFK activity cannot simply be selected.The RFK steadystate parameters suggest that the T203D and N210D may be used for the subsequent genomic mutations (Table 4).Therefore, we chose the two mutation sites to complete mutations on the R2 genome to study the effects of different mutations on RF production and strain growth.

Replacement of native ribF with mutant ribF through CRISPR/Cas9
The positive colonies were screened by colony PCR with ribC(F)-JC-F/R as primers, and positive clones yielded a 1080 bp band, while negative clones yielded a 1008 bp band (Supplementary Fig. S4).The positive colonies were further sequenced by Sangon Biotech (Shanghai, China).
The primers Mid(F)-F/R were used to amplify ribF M -T203D and ribF M -N210D to obtain the nucleotide fragments Mid(F)203 and Mid(F) 210.DNA repair templates were obtained by using primers Up(F), Down (F), and ribF M -T203D/ribF M -N210D as templates.After CRISPR/Cas9 genomic editing, the positive colonies were screened by sequencing, and designated as R203 and R210, respectively.

Effect of genomic ribF mutations on RFK activity and RF production
The RFK activity of different mutated strains is shown in Table 5.The most dramatic decrease in RFK activity was observed in strain R210, with a 78.48 % reduction.The same mutation on CaFADS was also observed with an 89.57% reduction [4].Compared to other strains (R2 and R203), the growth rate of R210 was severely impaired, and it was easy to understand that RFK reduction affected normal physiological functions because FMN and FAD were key cofactors.
To determine the ability of the mutated strains to produce RF, pNEW-AZ was transformed into strains R203 and R210 to generate engineered strains R6 and R7, respectively.As with strain R210, the growth rate of strain R7 was also inhibited.FMN and FAD are hydrophilic and do not pass the plasma membrane, and both of these RF derivates might be hydrolyzed to form RF and then transform into cells from LB medium [33].To restore the growth of the engineered strain R7 and facilitate the production of RF, RF at 20 mg/L was added to the LB medium to compensate for the lack of FMN/FAD before cumate induction.After 72 h of fermentation in LB medium with 10 g/L glucose (containing 50 μg/L kanamycin), the RF titers of different engineered strains were determined, and are shown in Table 6.R6 produced 657.38 ± 47.48 mg/L RF, with a yield of 72.30 ± 5.21 mg RF/g glucose, and with a 21.69 % increase compared to R5. R7 produced only 94.27 ± 0.75 mg/L RF.Meanwhile, although RF addition enhanced strain growth to some extent, the growth rate was still slower than that of the other strains.Flavoproteins with FMN/FAD as coenzymes are involved in the dehydrogenation of a variety of metabolites, in one-and two-electron transfer from and to redox centers, and in the activation of oxygen for oxidation and hydroxylation reactions [34].The N210D mutation severely decreased RFK activity, and the lack of FMN/FAD largely affected the physiological functions of the engineered strain and thus the synthesis of    RF, even when supplemented with RF in medium.

Discussion
In recent years, because RF production by microbial fermentation is more economical and environmentally friendly than chemical and semichemical synthesis, several RF production strains, which are mainly based on the use of B. subtilis, A. gossypii, Candida famata, and E. coli, have been developed and applied on an industrial scale to obtain RF [35,36].As a model organism, E. coli has the advantages of a clear metabolic background, rapid growth, low maintenance metabolism, and mature molecular tools suitable for its genetic manipulation, especially the ability of E. coli BL21 (DE3) to accumulate RF.The T7 expression system is widely used to express target proteins and obtain target products.However, its expensive inducer IPTG limits its application in large-scale fermentation [37].In addition, it suffers from severe leaky expression that may affect the yield of the target product [38,39].The T7 terminator that comes with the pET series of plasmids has only 74 % termination efficiency, resulting in read-through [40].This read-through can affect the expression of downstream elements such as antibiotic resistance genes, plasmid copy number control elements, or repressor proteins used to lower basal expression levels [41].Cumate is nontoxic to the host, inexpensive, and a carbon source-independent inducer, which provides an economical option for the large-scale production of valuable proteins and chemicals [37].To reduce leakage expression as well as realize large-scale production of RF at a later stage, we inserted six key genes of RF synthesis into the pNEW vector.The R5 strain transformed with pNEW-AZ showed a 10.61 % increase in RF production compared to the previous R4 strain based on the pET system for RF production.This result suggests that the cumate-induced expression system can be effectively applied in the production of target products, including RF.In the present study, it was found that although cumate is nontoxic, too high concentration apparently lowers the pH of the medium, which in turn has some negative effects on the fermentation, especially on the growth rate of the organisms.
The main function of prokaryotic FADS is to provide flavin cofactors and, at the same time, to maintain intracellular flavin and flavoprotein homeostasis [42].Different sources of FADS differ in their enzymatic properties, particularly CaFADS, and the differences are mainly in the catalytic efficiency of the enzyme (including RFK, FMNAT, or FADpp), whether it produces substrate or product inhibition, the redox environment required to produce enzymatic activity, the arrangement of the active center, and the way the protein polymerizes in solution [24,26].These differences allow FADS to effectively regulate intracellular flavin homeostasis in different cells.
Both I204 and H258 were involved in the formation of the important protein-ligand interactions, leading to the formation of the hydrogen bond and Pi electron clouds.In EcFADS, H258 was not only connected to the substrate ATP through hydrogen bonding at the active center, but also participated in the formation of the substrate RF active center through hydrophobic interaction.Thus, mutations of H258A and H258D resulted in a complete loss of RFK enzyme activity.The mutation in R303 affects the α-helix structure, resulting in large changes in the protein conformation and consequent reduction or loss of the RFK enzyme activity.The N210D mutation can disrupt the β-fold motif in the main structure of the protein, resulting in a change in the conformation of protein which makes the RFK activity decreased dramatically.The T203A mutation greatly enhances the RFK enzyme activity.Since alanine is more hydrophobic than threonine, the mutant in this locus promotes the formation of hydrophobic forces, which in turn enhances the RFK enzyme activity.L260, D261, and E299 are located in random coiling.Therefore, the associated mutations have little effect on the overall protein structure as well as the RFK activity.
Asn and Thr residues play an important role in coordinating cations at the active sites of kinases [3].In the study of RFK activity of the mutated proteins, the mutation of N210D greatly reduced RFK activity, which not only affects the growth of the strain, but also further affects RF production.The mutation of T203A enhanced the RFK activity by 103.11 %, whereas the mutation of T203D decreased the RFK activity by 29.30 %.In CaFADS, T208 and N210 provide the RFK active-site geometry for binding and catalysis, and mutations at these two sites substantially reduce the RFK activity and modulate the binding parameters at the FMNAT active site of CaFADS, altering the catalytic efficiency in the transformation of FMN into FAD [4].FMNAT activity was not measured in our study, and it is not known whether mutations in amino acids in the C-terminal RFK module of EcFADS will affect the FMNAT module.Lin et al. [21] reduced the ribF expression level by 35.17 % and elevated RF production by 77.05 % by replacing native RBS.Hu et al. [13] knocked down the expression of the ribF by synthetic regulatory small RNA, which enhanced RF production by up to 132.02 % compared to the basic strain WY0S.In contrast to the results of this study, mutation of genomic T203D reduced RFK enzyme activity by 24.27 %, while RF production was elevated by 21.69 %, and the RF titer reached 657.38 ± 47.48 mg/L.The first two operations regulate the entire FADS bifunctional enzyme activity, while the mutation in the C-RFK module in this study regulates mainly RFK activity.This may explain why the first two operations are more likely to increase RF production.Xu et al. [16] constructed an engineered strain RF18S that could produce 387.6 mg/L RF with 10 g/L glucose.Another strain LS02T was constructed by Liu et al. [14] which produced 667 mg/L of RF when cultivated in MSY medium supplied with 10 g/L glucose.The strain E. coli RF05S could produce 585.2 ± 13.6 mg/L RF [21].Compared to these strains, our constructed strain in this study produced 657.38 ± 47.48 mg/L RF.Thus, our study might provide a basis for the future industrial production of RF.

Conclusion
In summary, the vector pNEW-AZ constructed on the basis of the pNEW plasmid was able to enhance RF yield and reduce the inducer cost.We identified nine possible key amino acid residues in the RFK activity center of EcFADS by homology matching and molecular docking, selected two amino acid sites by RFK activity assay, and obtained two genetically engineered bacteria by subsequent CRISPR gene editing based on the modified R2 strain.After transformation of the plasmid pNEW-AZ, fermentation in LB medium containing 10 g/L glucose yielded 657.38 ± 47.48 mg/L RF, which was elevated by 34.60 % compared to R4.In addition, the T203A mutation greatly enhances RFK activity and can be used for later modification of FAN/FAD-producing strains.Although there have been studies on enhancing RF production by reducing the enzymatic activity of FADS and on the key site of the RFK active center, the present study is, to the best of our knowledge, the first to target mutations in key amino acid residues of the C-terminal RFK module of EcFADS to enhance RF production.

Data availability
The supplementary figures are available at the end of this manuscript.

CRediT authorship contribution statement
Bing Fu: conducted the experiments.Meng Chen: conducted the

Fig. 1 .
Fig. 1.Diagrammatic representation of the successive conversion of riboflavin into FMN and FAD.

B
.Fu et al.
R GGCTTCAGTTTTGAAGTCCATGGTCAGGTAC Note: Underlined letters indicate the restriction endonuclease-digested sites.B. Fu et al.
• C overnight.The single colony was selected and cultured in 5 mL LB with 50 μg/L spectinomycin.The plasmids were extracted and sequenced by Shanghai Biotech.The primers Up(C)-F/R and Down(C)-F/R were used to amplify the upstream and downstream homology arms [Up(C) & Down(C), ~500 bp] with the E. coli BL21(DE3) genome DNA as the template.The primers Mid(C)-F/R were used to obtain the nucleotide fragment ribC opt with the plasmid pET-3a(+)-ribC opt as the template.Overlap PCR was performed to acquire the donor DNA for repair, and the nucleotide fragments Up (C), Down(C), and ribC opt were used as templates, with Up(C)-F/Down (C)-R as primers.

Fig. 3 .
Fig. 3. Identification of key amino acid sites for RFK module of WT EcFADS.a. Result of RibF amino acid sequence alignment from four different microorganisms.b.A two-dimensional diagram of the molecular docking results of substrates (left: ATP; right: riboflavin) interaction with the RFK module of WT EcFADS.The hydrogen bonds between the amino acid and the substrate are represented with dotted lines (green).The hydrophobic bonds are represented with an eyelash shape.c.A three-dimensional diagram of the molecular docking results of substrates interaction with the RFK module of WT EcFADS.RF and ATP ligands are shown as sticks and colored with carbons in green and yellow, respectively.The Mg 2+ cation is shown as a yellow sphere.The FMNAT module is blue and the RFK module is pink.

Table 1
Strains and plasmids.

Table 3
Comparison of RFK activity of different mutant proteins.

Table 4
Steady-state kinetic parameters for the RFK activity of WT, T203D, and N210D.

Table 5
Comparison of RFK activities of different mutated strains.

Table 6
Comparison of fermentation parameters between R6 and R7 strains.Note: Letters indicate significant differences (P < 0.05).
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